1. Field of the Invention
The present invention relates generally to the fields of biochemistry and nucleic acid chemistry. More specifically, the present invention relates to in vitro selection of RNA nucleic acid binding species containing from one to three identical fluorescently labeled nucleotides.
2. Description of the Related Art
Nucleic acid binding species (aptamers) that bind a wide range of targets are readily selected from random sequence populations.3-6 Selected aptamers can recognize molecules as simple as amino acids7 or as complex as red blood cell membranes8. The incorporation of chemically modified bases into aptamers drastically improves their stabilities and potentially renders them suitable for use in homogenous assays with sera or urine samples.
Reagentless biosensors that can directly transduce molecular recognition to optical signals can potentiate the development of sensor arrays for a wide variety of analytes. Pluripotent nucleic acid binding species, aptamers, are readily selected, but can be difficult to adapt to biosensor applications. The adaptation of selected nucleic acid binding species (aptamers) to function as biosensors can further potentiate numerous diagnostic applications.1.2 
Some nascent examples of aptamer biosensors have already been developed. Fluorescently labeled aptamers and capillary electrophoresis coupled to laser induced fluorescence (CE-LIF) have been used to sensitively detect IgE and thrombin in solution. A labeled anti-thrombin aptamer immobilized on a glass support can detect thrombin in solution by following changes in evanescent-wave-induced fluorescence anisotropy10. Labeled anti-CD4 aptamers are used to stain mouse T cells that express human CD411. A labeled anti-human neutrophil elastase (HNE) aptamer is as effective as an anti-anti-human neutrophil elastase antibody for detecting human neutrophil elastase on beads12.
However, these analytical methods are essentially mimics of methods already developed with antibodies, and generally rely upon an indirect readout of binding following washing or other separation techniques. In contrast, molecules that can directly signal the presence of analytes are proving increasingly useful as biosensors13. For example, a mutant of the E. coli phosphate binding protein labeled with a fluorescent dye at the edge of its binding site14 exhibited a large increase in fluorescence upon inorganic phosphate-binding.15 A similarly labeled maltose binding protein quantitatively detects maltose in solution16, while a labeled glucose binding protein detects glucose17. The conjugation of both acceptor and donor fluorophores to cAMP-dependent protein kinase yielded a sensor in which fluorescence resonance energy transfer (FRET) is modulated by cAMP18.
Aptamers that bind small molecules have been shown to undergo conformational changes upon interactions with their cognate ligands19,20. A reporter fluorophore introduced into an aptamer in a region known to undergo conformational change can lead to a change in fluorescence intensity after the binding event. However, such an introduction of a fluorophore may result in a significant loss of binding energy, possibly due to steric hindrance or the perturbation of the conformational equilibrium.
Given that functional nucleic acids contain only four monomers with limited chemistries, and that nucleic acid structure is largely predicated on Watson-Crick pairs in which nucleotides are co-dependent, it is surprising that binding species and catalysts can b e selected from random sequence pools that are substantially depleted in a given nucleotide. However, Rogers and Joyce22 have shown that ribozymes lacking cytidine can be selected following continuous evolution of the Bartel Class I ligase. While aptamers and ribozymes can be selected from pools depleted in one of the nucleotides, a functional price is apparently paid for the lost chemistry and structure. The C-less ribozyme is roughly 100 to 10,000-fold slower than comparable ribozymes that contain cytidine.
It is advantageous, therefore, to reduce the apparent dissonance between ligand-binding and fluorescent signaling. A b initio selection methods that yield signaling aptamers containing only a few identical residues of a fluorescent nucleotide provide such a means. These selected signaling aptamers couple the broad molecular recognition properties of their aptamers with signal transduction.
The prior art is deficient in the lack of in vitro selection methods for signaling aptamers. The present invention fulfills this long-standing need and desire in the art.
The present invention provides a method of selecting signaling aptamers in vitro comprising the steps: synthesizing a DNA pool so that the DNA has a random insert of nucleotides in a specific skewed mole ratio; amplifying the DNA pool; transcribing an RNA pool from the amplified DNA wherein a nucleotide used in the RNA transcription is fluorescently labeled; applying the fluorescently labeled RNA pool to an affinity column wherein high-affinity fluorescent RNA molecules are removed from the fluorescently labeled RNA pool; obtaining a cDNA pool from the high-affinity fluorescent RNA molecules; repeating the amplification and selection steps on the fluorescent RNA molecules and cloning the fluorescent RNA molecules where the clones comprise signaling aptamers.
The present invention also provides a method of selecting signaling aptamers in vitro comprising the steps of synthesizing a DNA pool, where said DNA has a random insert of nucleotides and said nucleotides comprise a skewed mole ratio; amplifying the DNA pool wherein a nucleotide used in the DNA amplification is labeled with one or more reporter molecules; isolating the labeled single-stranded DNA from the amplified DNA; applying the labeled single-stranded DNA pool to an affinity column wherein high-affinity labeled DNA molecules are removed from the labeled DNA pool; repeating steps (a) through (d) so as to retain the high-affinity labeled DNA pool on the affinity column; and cloning the retained labeled DNA molecules where the clones comprise signaling aptamers.
In another embodiment of the present invention, there is provided a signaling aptamer that transduces the conformational change upon binding a ligand to a change in fluorescence intensity of a fluorescently labeled nucleotide incorporated into the RNA sequence of the signaling aptamer. Additionally, the fluorescently labeled nucleotide may be incorporated into a DNA signaling aptamer.
In a preferred embodiment of the present invention there is provided a fluoresceinated RNA anti-adenosine signaling aptamer.